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UNLABELLED The aim of this study was to simultaneously construct PCR-DGGE-based predictive models of Listeria monocytogenes and Vibrio parahaemolyticus on cooked shrimps at 4 and 10°C. Calibration curves were established to correlate peak density of DGGE bands with microbial counts. Microbial counts derived from PCR-DGGE and plate methods were fitted by Baranyi model to obtain molecular and traditional predictive models. For L. monocytogenes, growing at 4 and 10°C, molecular predictive models were constructed. It showed good evaluations of correlation coefficients (R(2) > 0.92), bias factors (Bf ) and accuracy factors (Af ) (1.0 ≤ Bf ≤ Af ≤ 1.1). Moreover, no significant difference was found between molecular and traditional predictive models when analysed on lag phase (λ), maximum growth rate (μmax ) and growth data (P > 0.05). But for V. parahaemolyticus, inactivated at 4 and 10°C, molecular models show significant difference when compared with traditional models. Taken together, these results suggest that PCR-DGGE based on DNA can be used to construct growth models, but it is inappropriate for inactivation models yet. This is the first report of developing PCR-DGGE to simultaneously construct multiple molecular models. SIGNIFICANCE AND IMPACT OF THE STUDY It has been known for a long time that microbial predictive models based on traditional plate methods are time-consuming and labour-intensive. Denaturing gradient gel electrophoresis (DGGE) has been widely used as a semiquantitative method to describe complex microbial community. In our study, we developed DGGE to quantify bacterial counts and simultaneously established two molecular predictive models to describe the growth and survival of two bacteria (Listeria monocytogenes and Vibrio parahaemolyticus) at 4 and 10°C. We demonstrated that PCR-DGGE could be used to construct growth models. This work provides a new approach to construct molecular predictive models and thereby facilitates predictive microbiology and QMRA (Quantitative Microbial Risk Assessment).

The growth process of Lactobacillus curvatus colonies was quantified by a coupled growth and diffusion equation incorporating a volumetric rate of lactic acid production. Analytical solutions were compared to numerical ones, and both were able to predict the onset of interaction well. The derived analytical solution modeled the lactic acid concentration profile as a function of the diffusion coefficient, colony radius, and volumetric production rate. Interaction was assumed to occur when the volume-averaged specific growth rate of the cells in a colony was 90% of the initial maximum rate. Growth of L. curvatus in solid medium is dependent on the number of cells in a colony. In colonies with populations of fewer than 10 5 cells, mass transfer limitation is not significant for the growth process. When the initial inoculation density is relatively high, colonies are not able to grow to these sizes and growth approaches that of broth cultures (negligible mass transfer limitation). In foods, which resemble the model solid system and in which the initial inoculation density is high, it will be appropriate to use predictive models of broth cultures to estimate growth. For a very low initial inoculation density, large colonies can develop that will start to deviate from growth in broth cultures, but only after large outgrowth.

Microbial interaction can be ignored in predictive microbiology under most conditions. We show that interactions are only important at high population densities, using published data on inhibition of growth of Listeria monocytogenes in broth. Our analysis using growth models from predictive microbiology indicated that interactions only occur at population densities of ∼108 cfu/ml of the protective cultures. Spoilage is evident at these levels, except for fermented foods. In bacterial colonies, diffusion limitation acts as a constraint to growth. We have shown that these constraints only become important after large outgrowth of colonies (in the order of 5-log growth in Lactobacillus curvatus colonies), which depends on the initial inoculation density. Intra-colony interactions play an important role under these conditions. There is no large outgrowth of colonies when the initial inoculation densities are high and broth culture growth can be used to approximate colony growth. © 2002 Elsevier Science B.V. All rights reserved. Chemicals/CAS: Culture Media

Fluorescence ratio imaging is a non-invasive technique for studying the formation of microgradients in immobilised bacterial colonies. These gradients can be quantified easily when combined with the gel cassette system designed at the Institute of Food Research, Norwich, UK. Colonies of Lactobacillus curvatus were observed using this technique and relevant pH gradients were present when the colonies reached a diameter of about 100 μm. These pH gradients were due to production of lactic acid by L. curvatus cells in the colonies. The spatial resolution of the images was about 1.5 μm (scale of bacterial cells) and therefore very suitable for observing local effects in colonies which ranged in sizes from 1 to 500 μm. Copyright (C) 2000 Elsevier Science B.V.